1. Field of the Invention
The present invention relates to a method of introducing at least one gene encoding a product into at least one cell of a connective tissue of a mammalian host for use in treating the mammalian host. This method discloses employing DNA vector molecules containing a gene encoding the product and infecting the connective tissue cells of the mammalian host using the DNA vector molecule. This invention provides a method of introducing at least one gene encoding a product into at least one cell of a connective tissue of a mammalian host for use in treating the mammalian host including employing non-viral means for effecting such introduction.
The present invention discloses ex vivo and in vivo techniques for delivery of a DNA sequence of interest to the connective tissue cells of the mammalian host. The ex vivo technique involves prior removal and culture of target autologous connective tissue cells, in vitro infection of the DNA sequence, DNA vector or other delivery vehicle of interest into the connective tissue cells, followed by transplantation to the modified connective tissue cells to the target joint of the mammalian host, so as to effect in vivo expression of the gene product of interest. The in vivo technique bypasses the requirement for in vitro culture of target connective tissues cells; instead relying on direct transplantation of the DNA sequence, DNA vector or other delivery vehicle to the target in vivo connective tissue cells, thus effecting expression of the gene product of interest.
The present invention also relates to a method to produce an animal model for the study of connective tissue pathologies and systemic indices of inflammation.
The present invention further relates to a method of using a gene encoding a truncated interleukin-1 receptor to resist the deleterious pathological changes associated with arthritis. More specifically, this invention provides a method wherein a gene coding for an extracellular interleukin-1 binding domain of an interleukin-1 receptor is introduced into synovial cells of a mammalian host in vivo for neutralizing the destructive activity of interleukin-1 upon cartilage and other soft tissues. As an alternative, the patients own synovial cells are transduced in vitro and introduced back into the affected joint, using transplantation procedures such as for example, intra-articular injection.
As an alternative to the in vitro manipulation of synovia, the gene encoding the product of interest is introduced into liposomes and injected directly into the area of the joint, where the liposomes fuse with synovial cells, resulting in an in vivo gene transfer to synovial tissue. As an additional alternative to the in vitro manipulation of synovia, the gene encoding the product of interest is introduced into the area of the joint as naked DNA. The naked DNA enters the synovial cell, resulting in an in vivo gene transfer to synovial tissue.
As an another alternative, hematopoietic progenitor cells or the mature lymphoid or myeloid cells may be transfected in vitro, recovered and injected into the bone marrow or peripheral bloodstream of the patient using techniques known to the skilled artisan.
The present invention also relates to methods of using various DNA sequences disclosed throughout this specification to provide therapeutic treatment for damaged cartilage, particularly full thickness human articular cartilage defects, such as damaged articular cartilage surrounding any joint. More specifically, this invention further provides a method wherein a gene or DNA sequence encoding a biologically active fragment thereof is transferred into in vitro cultured chondrocytes, with the resulting transfected chondrocytes surgically introduced into the area of cartilage damage of the mammalian host as a composition comprising the transduced chondrocyte polulation along with a suitable scaffold, such as a collagen gel, so as to effect in vivo expression of the DNA sequence of interest. Such genes or DNA fragments include but will not be limited to sequences encoding a biologically active protein or fragment of an interleukin-1 receptor antagonist protein, extracellular interleukin-1 binding domain of an interleukin-1 receptor, TGF-xcex21, TGF-xcex22, TGF-xcex23, and IGF-1.
2. Brief Description of the Related Art
Arthritis involves inflammation of a joint that is usually accompanied by pain and frequently changes in structure. Arthritis may result from or be associated with a number of conditions including infection, immunological disturbances, trauma and degenerative joint diseases such as, for example, osteoarthritis. The biochemistry of cartilage degradation in joints and cellular changes have received considerable investigation.
In a healthy joint, cells in cartilage (chondrocytes) and the surrounding synovium (synoviocytes) are in a resting state. In this resting state, these cells secrete basal levels of prostaglandin E2 and various neutral proteinases, such as, for example, collagenase, gelatinase and stromelysin, with the ability to degrade cartilage. During the development of an arthritic condition, these cells become activated. In the activated state, synoviocytes and chondrocytes synthesize and secrete large amounts of prostaglandin E2 and neutral proteinases.
In efforts to identify pathophysiologically relevant cell activators, it has been known that the cytokine interleukin-1 activates chondrocytes and synoviocytes and induces cartilage breakdown in vitro and in vivo. Additionally, interleukin-1 is a growth factor for synoviocytes and promotes their synthesis of matrix, two properties suggesting the involvement of interleukin-1 in the synovial hypertrophy that accompanies arthritis. In contrast, interleukin-1 inhibits cartilaginous matrix synthesis by chondrocytes, thereby suppressing repair of cartilage. Interleukin-1 also induces bone resorption and thus may account for the loss of bone density seen in rheumatoid arthritis. Interleukin-1 is inflammatory, serves as a growth factor for lymphocytes, is a chemotactic factor and a possible activator of polymorphonuclear leukocytes (PMNs). When present in a sufficient concentration, interleukin-1 may cause fever, muscle wasting and sleepiness.
The major source of interleukin-1 in the joint is the synovium. Interleukin-1 is secreted by the resident synoviocytes, which are joined under inflammatory conditions by macrophages and other white blood cells.
Much attention has been devoted to the development of a class of agents identified as the xe2x80x9cNon-Steroidal Anti-Inflammatory Drugsxe2x80x9d (hereinafter xe2x80x9cNSAIDsxe2x80x9d). The NSAIDs inhibit cartilage synthesis and repair and control inflammation. The mechanism of action of the NSAIDs appears to be associated principally with the inhibition of prostaglandin synthesis in body tissues. Most of this development has involved the synthesis of better inhibitors of cyclo-oxygenase, a key enzyme that catalyzes the formation of prostaglandin precursors (endoperoxides) from arachidonic acid. The anti-inflammatory effect of the NSAIDs is thought to be due in part to inhibition of prostaglandin synthesis and release during inflammation. Prostaglandins are also believed to play a role in modulating the rate and extent of leukocyte infiltration during inflammation. The NSAIDs include, such as, for example, acetylsalicylic acid (aspirin), fenoprofen calcium (Nalfon(copyright) Pulvules(copyright), Dista Products Company), ibuprofen (Motrin(copyright), The Upjohn Company), and indomethacin (Indocin(copyright), Merck, Sharp and Dohme).
In contrast, the studies upon which the present invention is based show that production of the various neutral proteinases with the ability to degrade cartilage occurs even if prostaglandin synthesis is completely blocked.
Therapeutic intervention in arthritis is hindered by the inability to target drugs, such as the NSAIDs, to specific areas within a mammalian host, such as, for example a joint. Traditional routes of drug delivery, such as for example, oral, intravenous or intramuscular administration, depend upon vascular perfusion of the synovium to carry the drug to the joint. This is inefficient because transynovial transfer of small molecules from the synovial capillaries to the joint space occurs generally by passive diffusion. This diffusion is less efficient with increased size of the target molecule. Thus, the access of large drug molecules, for example, proteins, to the joint space is substantially restricted. Intra-articular injection of drugs circumvents those limitations; however, the half-life of drugs administered intra-articularly is generally short. Another disadvantage of intra-articular injection of drugs is that frequent repeated injections are necessary to obtain acceptable drug levels at the joint spaces for treating a chronic condition such as, for example, arthritis. Because therapeutic agents heretofore could not be selectively targeted to joints, it was necessary to expose the mammalian host to systemically high concentrations of drugs in order to achieve a sustained, intra-articular therapeutic dose. Exposure of non-target organs in this manner exacerbated the tendency of anti-arthritis drugs to produce serious side effects, such as for example, gastrointestinal upset and changes in the hematological, cardiovascular, hepatic and renal systems of the mammalian host.
It has been shown that genetic material can be introduced into mammalian cells by chemical or biologic means. Moreover, the introduced genetic material can be expressed so that high levels of a specific protein can be synthesized by the host cell. Cells retaining the introduced genetic material may include an antibiotic resistance gene thus providing a selectable marker for preferential growth of the transduced cell in the presence of the corresponding antibiotic. Chemical compounds for inhibiting the production of interleukin-1 are also known.
U.S. Pat. No. 4,778,806 discloses a method of inhibiting the production of interleukin-1 by monocytes and/or macrophages in a human by administering through the parenteral route a 2-2xe2x80x2-[1,3-propan-2-onediyl-bis(thio)] bis-1 H-imidazole or a pharmaceutically acceptable salt thereof. This patent discloses a chemical compound for inhibiting the production of interleukin-1. By contrast, in one embodiment of the present invention, gene therapy is employed that is capable of binding to and neutralizing interleukin-1.
U.S. Pat. No. 4,780,470 discloses a method of inhibiting the production of interleukin-1 by monocytes in a human by administering a 4,5-diaryl-2 (substituted) imidazole. This patent also discloses a chemical compound for inhibiting the production of interleukin-1.
U.S. Pat. No. 4,794,114 discloses a method of inhibiting the S-lipoxygenase pathway in a human by administering a diaryl-substituted imidazole fused to a thiazole, pyrrolidine or piperidine ring or a pharmaceutically acceptable salt thereof. This patent also discloses a chemical compound for inhibiting the production of interleukin-1.
U.S. Pat. No. 4,870,101 discloses a method for inhibiting the release of interleukin-1 and for alleviating interleukin-1 mediated conditions by administering an effective amount of a pharmaceutically acceptable anti-oxidant compound such as disulfiram, tetrakis [3-(2,6-di-tert-butyl-4-hydroxyphenyl) propionyloxy methyl] methane or 2,4-di-isobutyl-6-(N,N-dimethylamino methyl)-phenol. This patent discloses a chemical compound for inhibiting the release of interleukin-1.
U.S. Pat. No. 4,816,436 discloses a process for the use of interleukin-1 as an anti-arthritic agent. This patent states that interleukin-1, in association with a pharmaceutical carrier, may be administered by intra-articular injection for the treatment of arthritis or inflammation. In contrast, the present invention discloses a method of using and preparing a gene that is capable of binding to and neutralizing interleukin-1 as a method of resisting arthritis.
U.S. Pat. No. 4,935,343 discloses an immunoassay method for the detection of interleukin-1 beta that employs a monoclonal antibody that binds to interleukin-1 beta but does not bind to interleukin-1 alpha. This patent discloses that the monoclonal antibody binds to interleukin-1 beta and blocks the binding of interleukin-1 beta to interleukin-1 receptors, and thus blocking the biological activity of interleukin-1 beta. The monoclonal antibody disclosed in this patent may be obtained by production of an immunogen through genetic engineering using recombinant DNA technology. The immunogen is injected into a mouse and thereafter spleen cells of the mouse are immortalized by fusing the spleen cells with myeloma cells. The resulting cells include the hybrid continuous cell lines (hybridomas) that may be later screened for monoclonal antibodies. This patent states that the monoclonal antibodies of the invention may be used therapeutically, such as for example, in the immunization of a patient, or the monoclonal antibodies may be bound to a toxin to form an immunotoxin or to a radioactive material or drug to form a radio pharmaceutical or pharmaceutical.
U.S. Pat. No. 4,766,069 discloses a recombinant DNA cloning vehicle having a DNA sequence comprising the human interleukin-1 gene DNA sequence. This patent provides a process for preparing human interleukin-1 beta, and recovering the human interleukin-1 beta. This patent discloses use of interleukin-1 as an immunological reagent in humans because of its ability to stimulate T-cells and B-cells and increase immunoglobulin synthesis.
U.S. Pat. No. 4,396,601 discloses a method for providing mammalian hosts with additional genetic capability. This patent provides that host cells capable of regeneration are removed from the host and treated with genetic material including at least one marker which allows for selective advantage for the host cells in which the genetic material is capable of expression and replication. This patent states that the modified host cells are then returned to the host under regenerative conditions. In the present invention, genetic material may be directly introduced (a) into host cells in vivo or (b) into synoviocytes in vitro for subsequent transplantation back into the patient""s joints.
U.S. Pat. No. 4,968,607 discloses a DNA sequence encoding a mammalian interleukin-1 receptor protein which exhibits interleukin-1 binding activity.
U.S. Pat. No. 5,081,228 discloses a DNA sequence encoding both the murine and human interleukin-1 receptor. This patent also provides a process for the in vitro expression of said DNA sequences.
U.S. Pat. No. 5,180,812 discloses a substantially pure preparation of the human interleukin-1 receptor protein.
In spite of these prior art disclosures, there remains a very real and substantial need for a method of introducing at least one gene encoding a product into at least one cell of a connective tissue of a mammalian host in vitro, or alternatively in vivo, for use in treating the mammalian host. Further, there is a need for a process wherein a gene encoding a truncated interleukin-1 receptor is used to resist the deleterious pathological changes associated with arthritis. More specifically there is a need for such a process where a gene coding for the extracellular interleukin-1 binding domain of the interleukin-1 receptor, capable of binding to and neutralizing interleukin-1 is expressed in host synovial cells in vivo. There is also a need to utilize one or more additional DNA sequences for delivery to and expression of a protein or protein fragment within a target host connective tissue cell, such as a synovial cell, so as to effect a treatment of various joint pathologies and concomitant systemic indices of inflammation.
There is also a very real and substantial need to treat various mammalian cartilage defects, in particular human articular and meniscal cartilage defects.
Brittberg et al. (1994, New England Journal of Medicine 331(14):879-895) disclose transplantation of non-modified human autologous chondrocytes cultured in vitro to correct articular cartilage defects. A biopsy of healthy cartilage was removed by arthroscopy from the damaged knee, cultured in vitro and transplantated by injection into the damaged area. The injected chondrocytes were secured within the damaged portion of articular cartilage by suture of periosteal flap taken from the medial tibia. No genetic modification of the cultured chondrocytes was reported or suggested by the authors.
Grande et al. (1989, J. Orthopaedic Research Society 7:208-219) utilized a similar surgical technique to transplant in vitro cultured rabbit chondrocytes in an attempt to repair a full-thickness cartilage defect.
The various techniques disclosed to date to treat full-thickness cartilage defects have had variable and limited success. None of these studies adequately demonstrate repair of the damaged cartilage with tissue which was histologically, biochemically, and biomechanically identical to normal cartilage. Moreever, the long term result has been poor as the repair tissue is fibrocartilage. None of these numerous attempts to overcome this long standing problem address adequate and/or appropriate cytokine mediation during the repair process. Multiple cytokines, such as transforming growth factor-xcex21 (TGF-xcex21) and insulin-like growth factor-1 (IGF-1), play significant roles in promoting chondrocyte anabolism and inhibiting chondrocyte catabolism. The presence of one or more of these cytokines during repair may be the key to regenerating normal cartilage. However, sustained delivery of sufficient quantities of a cytokine(s) to transplanted cells bound within a three-dimensional cartilage matrix would be difficult and impractical using the methods described above.
The present invention has met the hereinbefore described need. A method of introducing at least one gene encoding a product into at least one cell of a connective tissue of a mammalian host for use in treating the mammalian host is provided for in the present invention. This method includes employing recombinant techniques to produce a DNA vector molecule containing the gene encoding for the product and infecting the connective tissue cell of the mammalian host using the DNA vector molecule containing the gene coding for the product. The DNA vector molecule can be any DNA molecule capable of being delivered and maintained within the target cell or tissue such that the gene encoding the product of interest can be stably expressed. The DNA vector molecule preferably utilized in the present invention is either a viral DNA vector molecule or a plasmid DNA viral molecule. This method preferably includes introducing the gene encoding the product into the cell of the mammalian connective tissue for a therapeutic use.
One ex vivo method of treating a connective tissue disorder disclosed throughout this specification comprises initially generating a recombinant viral vector which contains a DNA sequence encoding a protein or biologically active fragment thereof. This recombinant viral vector is then used to infect a population of in vitro cultured connective tissue cells, resulting in a population of transfected connective cells. These transfected connective tissue cells are then transplanted to a target joint space of a mammalian host, effecting subsequent expression of the protein or protein fragment within the joint space. Expression of this DNA sequence of interest is useful in substantially reducing at least one deleterious joint pathology or indicia of inflammation normally associated with a connective tissue disorder.
The connective tissue cells are selected from the group of connective tissue consisting of a synovium, a cartilage, a tendon and a ligament, preferably synovial cells.
It is also preferred that a retroviral vector, such as MFG, be utilized as the viral vector.
Another preferred step in this ex vivo method is transplantation of transduced synovial cells by intraarticular injection.
It will be understood by the artisan of ordinary skill that the preferred source of cells for treating a human patient are the patients own cells, such as autologous synovial cells.
More specifically, this method includes employing as the gene a gene capable of encoding at least one of the materials which is selected from the group which includes (a) a human interleukin-1 receptor antagonist protein, preferably MFG-IRAP, or a biologically active derivative or fragment thereof, (b) a Lac Z marker gene capable of encoding a beta-galactosidase protein or a biologically active derivative or fragment thereof, (c) a soluble interleukin-1 receptor protein or a biologically active derivative or fragment thereof, (d) a soluble TNF-xcex1 receptor protein or a biologically active derivative or fragment thereof; (e) a proteinase inhibitor, and (f) a cytokine, and employing as the viral vector at least one vector which is selected from the group which includes (a) a retroviral vector including at least one of the materials selected from the group which includes MFG and BAG, (b) an adeno-associated virus, (c) an adenovirus, and (d) a herpes virus, including but not limited to herpes simplex 1 or herpes simplex 2.
A further embodiment of the present invention includes employing as the gene a gene capable of encoding at least one of the materials which is selected from the group which includes (a) a human interleukin-1 receptor antagonist protein or a biologically active derivative or fragment thereof, (b) a Lac Z marker gene capable of encoding a beta-galactosidase protein or a biologically active derivative or fragment thereof, (c) a soluble interleukin-1 receptor protein or a biologically active derivative or fragment thereof, (d) a soluble TNF-xcex1 receptor protein or a biologically active derivative or fragment thereof; (e) a proteinase inhibitor, and (f) a cytokine, and employing as the DNA plasmid vector any DNA plasmid vector known to one of ordinary skill in the art capable of stable maintenance within the targeted cell or tissue upon delivery, regardless of the method of delivery utilized. One such method is the direct delivery of the DNA vector molecule, whether it be a viral or plasmid DNA vector molecule, to the target cell or tissue. This method also includes employing as the gene a gene capable of encoding at least one of the materials selected from the group which includes (a) a human interleukin-1 receptor antagonist protein or biologically active derivative or fragment thereof, (b) a Lac Z marker gene capable of encoding a beta-galactosidase protein or biologically active derivative or fragment thereof, (c) a soluble interleukin-1 receptor protein or biologically active derivative or fragment thereof, (d) a soluble TNF-xcex1 receptor protein or a biologically active derivative or fragment thereof; (e) a proteinase inhibitor, and (f) a cytokine. In a specific method disclosed as an example, and not as a limitation to the present invention, a DNA plasmid vector containing the interleukin-1 beta (IL-1xcex2) coding sequence was ligated downstream of the cytomegalovirus (CMV) promoter. This DNA plasmid construction was encapsulated within liposomes and injected intra-articularly into the knee joints of recipient rabbits. IL-1xcex2 was expressed and significant amounts of interleukin-1 beta was recovered from the synovial tissue. An alternative is injection of the naked plasmid DNA into the knee joint, allowing direct transfection of the DNA into the synovial tissue. Injection of IL-1xcex2 into the joint of a mammalian host allows for prolonged study of various joint pathologies and systemic indices of inflammation, as described within this specification.
Another embodiment of this invention provides a method for introducing at least one gene encoding a product into at least one cell of a connective tissue of a mammalian host for use in treating the mammalian host. This method includes employing non-viral means for introducing the gene encoding for the product into the connective tissue cell. More specifically, this method includes employing non-viral means which is selected from at least one of the group which includes (a) at least one liposome, (b) Ca3(PO4)2, (c) electroporation, and (d) DEAE-dextran, and includes employing as the gene a gene capable of encoding at least one of the materials selected from the group which includes (a) a human interleukin-1 receptor antagonist protein or biologically active derivative or fragment thereof, (b) a Lac Z marker gene capable of encoding a beta-galactosidase protein or biologically active derivative or fragment thereof, (c) a soluble interleukin-1 receptor protein or biologically active derivative or fragment thereof, (d) a soluble TNF-xcex1 receptor protein or a biologically active derivative or fragment thereof; (e) a proteinase inhibitor, and (f) a cytokine.
A further embodiment of this invention provides an additional method for introducing at least one gene encoding a product into at least one cell of a connective tissue of a mammalian host for use in treating the mammalian host. This additional method includes employing the biologic means of utilizing a virus to deliver the DNA vector molecule to the target cell or tissue. Preferably, the virus is a psuedovirus, the genome having been altered such that the psuedovirus is capable only of delivery and stable maintenance within the target cell; but not retaining an ability to replicate within the target cell or tissue. The altered viral genome is further manipulated by recombinant DNA techniques such that the viral genome acts as a DNA vector molecule which contains the heterologous gene of interest to be expressed within the target cell or tissue. This method also includes employing as the gene a gene capable of encoding at least one of the materials selected from the group which includes (a) a human interleukin-1 receptor antagonist protein or biologically active derivative or fragment thereof, (b) a Lac Z marker gene capable of encoding a beta-galactosidase protein or biologically active derivative or fragment thereof, (c) a soluble interleukin-1 receptor protein or biologically active derivative or fragment thereof, (d) a soluble TNF-xcex1 receptor protein or a biologically active derivative or fragment thereof, (e) a proteinase inhibitor, and (f) a cytokine.
A further embodiment of this invention provides for an animal model to study connective tissue pathologies and indices of systemic inflammation. This model utilizes either ex vivo or in vivo delivery of at least one gene or DNA sequence of interest encoding a product into a least one cell of a connective tissue of a mammalian host. Examples of joint pathologies which can be studied in the present invention include, but are by no means limited to, joint pathologies such as leukocytosis, synovitis, cartilage breakdown and suppression of cartilage matrix synthesis. Examples of indices of systemic inflammation which include, but are by no means limited to, erythrocyte sedimentation rate, fever and weight loss.
An embodiment of the present invention is a method to produce an animal model for the study of joint pathologies. This embodiment comprises generating a recombinant viral vector which contains a DNA sequence encoding a protein or biologically active fragment thereof, infecting a population of in vitro cultured connective tissue cells with said recombinant viral vector, resulting in a population of transfected connective cells, and transplanting said transfected connective cells to a joint space of a mammalian host. This method will allow for collection of data regarding the effect of various expressed proteins or protein fragment on various deleterious joint pathologies or indicia of inflammation normally associated with a connective tissue disorder.
The connective tissue cells from this embodiment are also selected from the group of connective tissue consisting of a synovium, a cartilage, a tendon and a ligament, preferably synovial cells, including but not limited to autologous cells removed directly from the mammalian host of which the target joint resides.
A preferable mode of introducing transduced synovial cells to the joint space is by intraarticular injection.
A preferable mode of introducing transduced chrondrocyte cells to the area of the targeted cartilage defect is by surgical implantation.
A DNA sequence exemplified for animal model studies is a DNA sequence encoding encoding human IL-1xcex1, human IL-xcex2, or a biologically active fragment thereof.
Another DNA sequence exemplified for animal model studies is a DNA sequence encoding human tumor necrosis factor-a or a biologically active fragment thereof.
Another embodiment of a method to produce an animal model for the study of joint pathologies utilizes a recombinant DNA plasmid vector, which contains the DNA sequence of interest encoding a protein or biologically active fragment thereof. This recombinant DNA plasmid vector is used to transform a population of in vitro cultured connective tissue cells. The transformed connective cells, preferably synovial cells, are transplanted to a joint space of a mammalian host, so as to provide data regarding various joint pathologies and systemic indices of inflammation associated with connective tissue disorders.
This particular embodiment is exemplified by the ex vivo based delivery of MFG-IL-1xcex2 to a target rabbit knee joint, causing various joint pathologies and systemic indices of inflammations.
Another exemplification of this particular embodiment of the present invention is delivery of the CMV-IL-1xcex2 plasmid construction to the rabbit knee joint via liposome-mediated delivery.
An animal model as described and exemplified in this specification measures the ability of various gene therapy applications disclosed throughout this specification to withstand challenges from known causative agents (such as IL-1xcex2) of joint pathologies and inflammatory side effects.
An additional embodiment of the present invention relates to a method of using a DNA sequence encoding a biologically active interleukin-1 receptor antagonist (IRAP) or portion thereof for treatment of connective tissue joint pathologies. The DNA sequence encoding IRAP or a biologically active fragment thereof may be delivered to the connective tissue of a mammalian host by any combination of various vector strategies and transduction techniques disclosed throughout this specification.
A preferred method of the embodiment of delivering IRAP to a target joint space involves delivery of the IPAP gene to the synovial lining of a mammalian host through use of a retroviral vector with the ex vivo technique disclosed within this specification. In other words, a DNA sequence of interest encoding a functional IRAP protein or protein fragment is subcloned into a retroviral vector of choice, the recombinant viral vector is then grown to adequate titers and used to infect in vitro cultured synovial cells, and the transduced synovial cells, preferably autografted cells, are transplanted into the joint of interest, preferably by intra-articular injection.
Another preferred method of the present invention involves direct in vivo delivery of the IRAP gene to the synovial lining of a mammalian host through use of either an adenovirus vector, adeno-associated virus (AAV) vector or herpes-simples virus (HSV) vector. In other words, a DNA sequence of interest encoding a functional IRAP protein or protein fragment is subcloned into the respective viral vector, the IRAP containing viral vector is then grown to adequate titers and directed into the joint space, preferably by intra-articular injection. A retroviral-IRAP construct, such as MFG-IRAP may also be utilized to directly target previously inflamed connective tissue cells within the joint space.
Direct intra-articular injection of a DNA molecule containing the gene of interest into the joint results in transfection of the recipient synovial cells and hence bypasses the requirement of removal, in vitro culturing, transfection, selection, as well as transplanting the DNA vector containingxe2x80x94synoviocytes (as disclosed in the Example section) to promote stable expression of the heterologous gene of interest. Methods of presenting the DNA molecule to the target connective tissue of the joint includes, but is not limited to, forming a complex of the DNA molecule with cationic liposomes, subcloning the DNA sequence of interest in a retroviral vector as described throughout this specification, or the direct injection of the DNA molecule itself into the joint. The DNA molecule, regardless of the form of presentation to the knee joint, is preferably presented as a DNA vector molecule, either as recombinant viral DNA vector molecule or a recombinant DNA plasmid vector molecule. Expression of the heterologous gene of interest is ensured by inserting a promoter fragment active in eukaryotic cells directly upstream of the coding region of the heterologous gene. One of ordinary skill in the art may utilize known strategies and techniques of vector construction to ensure appropriate levels of expression subsequent to entry of the DNA molecule into the synovial tissue. In vivo delivery of various viral and non-viral vectors to the rabbit knee joint are described in Example XIV.
Another embodiment of the present invention is delivery of a DNA sequence of interest to the connective tissue of a mammalian host by any of the methods disclosed within this specification so as to effect in vivo expression of the human TNF-xcex1 soluble receptor or a biologically active fragment thereof.
Another embodiment of the present invention is delivery of a DNA sequence of interest to the connective tissue of a mammalian host by any of the methods disclosed within this specification so as to effect in vivo expression of various cytokines which possess anti-inflammatory and immunomodulatory characteristics, including but by no means limited to interleukin-4, interleukin-10, interleukin-13 and viral interleukin-10 (vIL-10).
Another embodiment of the present invention is delivery of a DNA sequence of interest to the connective tissue of a mammalian host by any of the methods disclosed within this specification so as to effect in vivo expression of various antiadhesion molecules so as to inhibit cell-cell and cell-matrix interactions. Examples of such proteins or protein fragments include but are not limited to soluble ICAM-1 and soluble CD44.
Another embodiment of the present invention is delivery of a DNA sequence of interest to the connective tissue of a mammalian host by any of the methods disclosed within this specification so as to effect in vivo expression of various cartilage growth factors, including but not limited to IGF-1, TGF-xcex21, TGF-xcex22, and TGF-xcex23.
Another embodiment of the present invention is delivery of a DNA sequence of interest to the connective tissue of a mammalian host by any of the methods disclosed within this specification so as to effect in vivo expression of various free radical antagonists, thus preventing the deleterious effects of free radical formation within the afflicted joint. Examples include but are not limited to the superoxide dismutase and proteins or protein fragments which inhibit NO.
Another embodiment of the present invention regarding delivery of the IRAP gene to the synovial lining of a mammalian host involves use subcloning this DNA sequence of interest into a viral vector such as adenovirus, adeno-associated virus and herpes-simplex virus. The respective recombinant IRAP based viral vector is then delivered to the joint by direct in vivo injection so as to effect in vivo expression of the IRAP protein or biologically active fragment thereof.
Another embodiment of this invention provides a method of using the gene encoding an extracellular interleukin-1 binding domain of the interleukin-1 receptor. This gene is capable of binding to and neutralizing interleukin-1 in vivo to substantially resist the degradation of cartilage in a mammalian host. Unlike previous pharmacological efforts, the method of this invention employs gene therapy in vivo to address the chronic debilitating effects of arthritis.
A preferred method of using the gene coding for the truncated interleukin-1 receptor of this invention involves employing recombinant techniques to generate a cell line which produces infectious retroviral particles containing the gene coding for the truncated interleukin-1 receptor. The producer cell line is generated by inserting the gene coding into a retroviral vector under the regulation of a suitable eukaryotic promoter, transfecting the retroviral vector containing the gene coding into the retroviral packaging cell line for the production of a viral particle that is capable of expressing the gene coding for the truncated interleukin-1 receptor, and infecting the synovial cells of a mammalian host using the viral particle.
More specifically, the method of using the hereinbefore described gene coding for the truncated interleukin-1 receptor involves introducing the viral particles obtained from the retroviral packaging cell line directly by intra-articular injection into a joint space of a mammalian host that is lined with synovial cells. In a preferred embodiment, synoviocytes recovered from the knee joint are cultured in vitro for subsequent utilization as a delivery system for gene therapy. It will be apparent that Applicants are not limited to the use of the specific synovial tissue disclosed. It would be possible to utilize other tissue sources, such as skin cells, for in vitro culture techniques. The method of using the gene of this invention may be employed both prophylactically and in the therapeutic treatment of arthritis. It will also be apparent that Applicants are not limited to prophylactic or therapeutic applications in treating only the knee joint. It would be possible to utilize the present invention either prophylactically or therapeutically to treat arthritis in any susceptible joint.
In another embodiment of this invention, a method of using the hereinbefore described gene coding for the truncated interleukin-1 receptor involves infecting synovial cells in culture with the viral particles and subsequently transplanting the infected synovial cells back into the joint. This method of using the gene of this invention may also be employed prophylactically and in the therapeutic treatment of arthritis in any area susceptible to the disorder.
In another embodiment of this invention, a method of using the gene coding for an extracellular interleukin-1 binding domain of the interleukin-1 receptor that is capable of binding to and neutralizing interleukin-1 includes employing recombinant techniques to produce a retrovirus vector carrying two genes. The first gene encodes the extracellular interleukin-1 binding domain of the interleukin receptor, and the second gene encodes for selectable antibiotic resistance. This method of use involves transfecting the retrovirus vector into a retrovirus packaging cell line to obtain a cell line producing infectious retroviral particles carrying the gene.
Another embodiment of this invention provides a method of preparing a gene encoding an extracellular interleukin-1 binding domain of the interleukin-1 receptor including synthesizing the gene by a polymerase chain reaction, introducing the amplified interleukin-1 receptor coding sequence into a retroviral vector, transfecting the retroviral vector into a retrovirus packaging cell line and collecting viral particles from the retrovirus packaging cell line.
In another embodiment of this invention, a compound for parenteral administration to a patient in a therapeutically effective amount is provided for that contains a gene encoding an extracellular interleukin-1 binding domain of the interleukin-1 receptor and a suitable pharmaceutical carrier.
Another embodiment of this invention provides for a compound for parenteral administration to a patient in a prophylactically effective amount that includes a gene encoding an extracellular interleukin-1 binding domain of the interleukin-1 receptor and a suitable pharmaceutical carrier.
An additional embodiment of the invention involves transfection of hematopoietic progenitor cells or mature lymphoid or myeloid cells with a DNA vector molecule containing any of the gene or genes disclosed throughout the specification. The transfected cells are recovered and injected into the bone marrow marrow or peripheral bloodstream of the patient using techniques known to the skilled artisan. It will be possible, within the scope of this method, to use cells derived from donor bone marrow instead of cells derived from recipient bone marrow so as to modify rejection.
The present invention also relates to methods of using various DNA sequences disclosed throughout this specification to provide therapeutic treatment for damaged cartilage, particularly damaged articular cartilage, such as articular cartilage within the human knee joint. For example, the present invention provides for methods of treating damaged or defective articular cartilage by introducing a DNA sequence into chondrocytes whereby expression of the DNA sequence of interest in vivo provides therapeutic relief from cartilage defects.
The present invention provides gene therapy methods for delivery of DNA sequences of interest to chondrocyte cells cultured in vitro and transplantation of these transfected cells to the damaged articular cartilage within a mammalian host. Such DNA sequences which are utilized express proteins or biologically active fragments thereof which improve or maintain chondrogenesis. Viral promoters active in eukaryotic cells, as well as the mixing and matching of these promoter and additional enhancer sequences may be utilized in practicing the claimed invention. Also, promoters useful in plasmid constructions, including but not limited to a cytomegalovirus (CMV) promoter, a Rous Sarcoma virus (RSV) promoter, a Murine Leukemia Virus (MLV) promoter, a xcex2-actin promoter, as well as any cell-specific eukaryotic promoter sequence that would be known to be active in the cell targeted for transduction. As an alternative, the present invention provides for use of alternative promoters, e.g., strong chondrocyte promoters such as the type II collagen gene promoter. Additionally, the present invention allows for use of inducible promoters, including but not limited to inducible promoters regulating expression of IL-1, IL-6 and IL-8. Any eukaryotic promoter and/or enhancer sequence available to the skilled artisan which is known to control expression of the nucleic acid of interest may be used in either a viral or plasmid vector construction. As mentioned above, other promoters and vector constructs may be utilized to either shorten or lengthen the duration of in vivo expression within the transplanted chondrocyte/scaffold matrix.
In a specific embodiment of the present invention, a method of treating a mammalian cartilage defect is disclosed which comprises generating a recombinant viral vector containing a DNA sequence expressing a protein or biologically active fragment thereof, infecting a population of in vitro cultured chondrocyte cells with the recombinant viral vector so as to generate a population of transfected chondrocyte cells. These transfected chondrocyte cells are then transplanted to the joint area containing the damaged articular cartilage where expression of the recombinant DNA sequence provides therapeutic relief.
In a preferred embodiment of the present invention, the chondrocyte cells retrieved for in vitro culture prior to transfection and transplantation are autologous cells.
In a particular embodiment of the present invention, cultures of articular chondrocytes to be used for allotransplantation are either transduced with the recombinant viral or plasmid DNA vector and selected with G418. Confluent monolayers of chondrocytes are harvested washed, and counted. These chondrocytes are added to a collagen solution, which is allowed to gel prior to transplantation. The chondrocytelcollagen mixture is adhered to the damaged region of articular cartilage with fibrin glue, a mixture of fibrinogen and thrombin.
Any of the vector and/or genes disclosed throughout this specification possess the potential for therapeutic use in chondrocyte-based applications. Additionally, any such vectors and/or gene disclosed within the specification may be used in a model animal system to monitor, for example, localized effects of continuous cytokine expression in cartilage formation and rehabilitation. Such preferred vectors include, but are not limited to, a retroviral vector, such as MFG or BAG, and any plasmid DNA construct as disclosed throughout this specification. Preferred genes of biologically active gene fragments include but are not limited to human transforming growth factor-xcex21 (TGF-xcex21), human transforming growth factor-xcex22 (TGF-xcex22), human transforming growth factor-xcex23 (TGF-xcex23), insulin-like growth factor-1 (IGF-1), bone morphogenetic proteins (BMPs), IRAP and the extracellular domain of the interleulin-1 receptor protein.
Another preferred method of the present invention involves non-viral based delivery of the DNA sequence of interest to the in vitro cultured, preferably utilizing a plasmid DNA vector, as discussed within this specification. Therefore, the invention also provides for treatment of a mammalian cartilage defect which comprises generating a recombinant plasmid DNA vector which contains a DNA sequence encoding a protein or biologically active fragment thereof, infecting a population of in vitro cultured chondrocyte cells with the recombinant viral vector so as to generate a population of transfected chondrocyte cells. These transfected chondrocyte cells are then transplanted to the joint area containing the damaged articular cartilage where expression of the recombinant DNA sequence provides therapeutic.
To this end, as discussed throughout this specification, the present invention also provides for the use of non-viral mediated delivery systems to chondrocytes cultured in vitro, including, but not limited to (a) direct injection of naked DNA; (b) liposome mediated transduction; (c) calcium phosphate [Ca3(PO4)2] mediated cell transfection, the genetically transformed cells then returned extraarticularly to the mammalian host; (d) mammalian host cell transfection by electroporation, the genetically transformed cells then returned extraarticularly to the mammalian host; (e) DEAE-dextran mediated cell transfection, the genetically transformed cells then returned extraarticularly to the mammalian host; (f) polybrene mediated delivery; (g) protoplast fusion; (h) microinjection; and (i) polylysine mediated transformation.
The specification enables gene delivery and expression to both synovial cells and chondrocyte cells, each a respective connective tissue. The advantages of both direct in vivo and ex vivo methods of delivery are described in this specification. To this end, the present invention also teaches a combinatorial use of synovial and chondrocyte cell delivery methods which provide prophylactic or therapeutic relief from various joint pathologies enumerated throughout the specification.
One or more distinct DNA sequences can be delivered to the effected joint or joints by using a strategy whereby multiple DNA sequences, each housed within an appropriate recombinant vector, is transferred to chondrocyte cells and/or synovial cells by the methods disclosed throughout the specification. It is then possible to deliver gene or gene fragment combinations which will promote either a prophylactic or therapeutic response in vivo.
It is preferred that the ex vivo method described above for gene transfer to chondrocytes be utilized in conjunction with ex vivo method of gene transfer to synovial cells.
It is also preferred that the ex vivo method described above for gene transfer to chondrocytes be utilized in conjunction with direct ex vivo method of gene transfer to synovial cells.
Therefore, a method of treating a human full-thickness mammalian cartilage defect is disclosed which involves infecting a population of in vitro cultured autologous chondrocyte cells with at least a first recombinant viral vector containing a DNA sequence encoding a protein or biologically active fragment which results in a population of transfected chondrocyte cells, infecting a population of in vitro cultured autologous synovial cells with at least a second recombinant viral vector containing a DNA sequence encoding a protein or biologically active fragment which results in a population of transfected synovial cells, and transplanting the transfected chondrocyte cells and synovial cells to the appropriate joint space as described throughout this specification such that subsequent expression the recombinant proteins within the targeted joint space substantially alleviates the cartilage defect.
In a preferred embodiment of dual gene transfer delivery methods, the transfected synovial cells are introduced into the joint space by intra-articular injection.
In a preferred embodiment of dual gene transfer, one DNA sequence is subcloned into a recombinant vector and targeted to the joint space by synovial cell transfection and intra-articular injection, wherein a second DNA sequence is subcloned into a recombinant vector in a second procedure and targeted to the area of damaged articular cartilage.
The methods of the present invention are tailored primarily for treatment of genetic based connective tissue diseases or disorders. However, it will be known upon review of this specification that the methods of the present invention may also be utilized for treating injuries of the type encountered by sports medicine orthopaedists.
It is an object of the present invention to provide a method of introducing at least one gene encoding a product into at least one cell of a connective tissue of a mammalian host for use in treating the mammalian host.
It is an object of the invention to provide a method of introducing a gene encoding a product into at least one cell of a connective tissue of a mammalian host for a therapeutic use.
It is an object of the present invention to provide a method of introducing into the synovial lining cells of a mammalian arthritic joint at least one gene which codes for proteins having therapeutic properties.
It is an object of the present invention to provide an animal model for the study of connective tissue pathology.
It is an object of the present invention to provide a method of using in vivo a gene coding for the extracellular interleukin-1 binding domain of the interleukin-1 receptor that is capable of binding to and neutralizing substantially all isoforms of interleukin-1, including interleukin-1 alpha and interleukin-1 beta.
It is an object of the present invention to provide a method of using in vivo a gene coding for IRAP or a biologically active derivative thereof which is a competitive inhibitor of and therefore substantially neutralizes all isoforms of interleukin-1, including interleukin-1 alpha and interleukin-1 beta.
It is an object of the present invention to provide a method of using a gene in vivo in a mammalian host that is capable of binding to and neutralizing substantially all isoforms of interleukin-1 and thus, substantially resist the degradation of cartilage and protect surrounding soft tissues of the joint space.
It is an object of the present invention to provide a method of using in vivo a gene coding for the extracellular interleukin-1 binding domain of the interleukin-1 receptor that is capable of binding to and neutralizing substantially all isoforms of interleukin-1 for the prevention of arthritis in patients that demonstrate a high susceptibility for developing the disease.
It is an object of the present invention to provide a method of using in vivo a gene coding for IRAP that is capable of acting as a competitive inhibitor of and therefore substantially neutralizes all isoforms of interleukin-1 for the prevention of arthritis in patients that demonstrate a high susceptibility for developing the disease.
It is an object of the present invention to provide a method of using in vivo a gene coding for an extracellular interleukin-1 binding domain of an interleukin-1 receptor that is capable of binding to and neutralizing substantially all isoforms of interleukin-1 for the treatment of patients with arthritis.
It is an object of the present invention to provide a method of using in vivo a gene coding for IRAP or a biologically active derivative thereof which is a competitive inhibitor of and therefore substantially neutralizes all isoforms of interleukin-1 for the treatment of patients with arthritis.
It is an object of the present invention to provide a method of using in vivo a gene or genes that address the chronic debilitating pathophysiology of arthritis.
It is a further object of the present invention to provide a compound for parenteral administration to a patient which comprises a gene encoding an extracellular interleukin-l binding domain of the interleukin-1 receptor and a suitable pharmaceutical carrier.
It is a further object of the present invention to provide a compound for parenteral administration to a patient which comprises a gene encoding IRAP and a suitable pharmaceutical carrier.
It is an object of the present invention to provide a gene therapy based method of treating articular cartilage defects which involves transfecting cultured chondrocytes with a recombinant vector expressing a protein or protein fragment and transplanting these genetically modified chondrocytes to the location of the articular cartilage defect.
It is also an object of the present invention to utilize gene transfer to both synovial cells and chondrocyte cells and the subsequent methods of joint space delivery to treat the identical malady.
It is also an object of the present invention to utilize the chondrocyte-based methods of gene transfer methods described in this specification for use in animal models.
These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.